Device and methods for liquid crystal-based bioagent detection

ABSTRACT

The present invention provides liquid crystal-based devices and methods for bioagent detection. In certain aspects, the present invention is directed to devices and methods utilizing liquid crystals and membranes containing polymerized targets that can report the presence of bioagents including, but not limited to, enzymes, antibodies, and toxins.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisionalapplication 60/731,824, filed Oct. 31, 2005, which is incorporatedherein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under N00014-04-1-0659awarded by the Navy/ONR. The government has certain rights in theinvention.

FIELD OF THE INVENTION

This invention relates generally to methods of analyte detection usingliquid crystals. In particular, the present invention is directed todevices and methods utilizing liquid crystals and membranes containingpolymerized targets that can report the presence of bioagents including,but not limited to, enzymes and antibodies.

BACKGROUND OF THE INVENTION

Most present biosensors take advantage of biologically active materialsfor high sensitivity and selectivity. In general, the biosensor includesa biorecognition structure (e.g., a membrane) in contact with orinterrogated by a transducer. The biologically active materialrecognizes a particular biological molecule through a reaction, specificadsorption, or other physical or chemical process, and the transducerconverts the output of this recognition into a usable signal, usuallyelectrical or optical. Many approaches have been explored to achieveultra-sensitive detection of bio-species. These biodetection approachescan be categorized as either an engineering-oriented approach or abiological-oriented approach. In other words, most biodetection schemesare either based on relatively complex electronic, photonic and/orelectrochemical methods or more elegant biomolecular methods (e.g.enzyme linked immunosorbent assay, or ELISA) typically with an opticalor spectrometry-based readout.

By way of example, one process utilizes photonics integrated on amicrochip to study the interaction between the optical field and thetarget bio-analyte. Because most biorecognition processes occur in anaqueous ambient, this approach requires the integration of photonics,highly sensitive microelectronics and microfluidic systems on a singlemicrochip. The use of ion-channel switches as biosensors has also beenexplored, but the bioelectronic interface is a delicate one. Often, whenan approach promises very high sensitivity, the output signal from thebiorecognition is very small, thus requiring extremely highly-sensitiveon-chip microelectronics for signal amplification, processing andwireless transmission. The high demand of these approaches on systemintegration and high sensitivity photonics and electronics circuitrypresents a big challenge to the biosensors in terms of cost, reliabilityand power consumption. The more biomolecular based approaches, likeELISA, are simple, but typically require a macro scale spectrometrysystem to quantify the output.

Therefore, it is a primary object and feature of the present inventionto provide a bioagent detection device that is highly sensitive andselective, has a quick response time, and generates few false alarms.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a liquid crystaldevice for detecting the presence of a bioagent in a sample. Such adevice includes: (a) a liquid crystal; (b) an aqueous phase positionedsuch that an interface exists between the liquid crystal and the aqueousphase; and (c) a membrane containing a polymerized target of a bioagent.The membrane is located at the interface between the liquid crystal andthe aqueous phase. Interaction of the bioagent with the polymerizedtarget causes an orientation change in the liquid crystal therebyindicating the presence of the bioagent in the sample.

In certain embodiments, the bioagent is an enzyme and the target is asubstrate for the enzyme. Preferred embodiments are directed to thedetection of proteases, metalloproteases, and, most preferably,neurotoxins with metalloprotease activities. Accordingly, embodimentsfor neurotoxin detection include a substrate that is a peptide cleavableby the respective neurotoxin. For example, certain of the presentdevices detect the presence of botulinum toxin using as substrate apeptide cleavable by botulinum toxin (e.g., synaptosomal-associatedprotein 25 (SNAP-25) or a cleavable fragment thereof). In otherembodiments, the enzyme detected is a neurotoxin with phospholipase A₂activity and the substrate is a 1,2-diacyl-3-sn-phosphoglyceridecontaining an sn-2 ester bond.

In yet other embodiments, the liquid crystal device detects the presenceof an antibody in a sample by using as target an antigen recognized bythe respective antibody. As well, a sample may contain an antigen as abioagent with the target then being an antibody that recognizes theantigen.

The polymerized target may be provided in a variety of forms but targetmolecules are preferably cross-linked by a cross-linking agent. Suitablecross-linking agents include, but not limited to, zero lengthcross-linkers (e.g., 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC)); homobifunctional cross-linkers (e.g., the NHSester dithiobis(succinimidylpropionate (DSP), the imidoester dimethyladipimidate (DMA), sulfhydryl-reactive1,4-Di-[3′-(2′-pyridyldithio)priopionamido]butane (DPDPB), thedifluorobenzene derivative 1,5-difluoro-2,4-dinitrobenzene (DFDNB),formaldehyde, bis-epoxides, and hydrazides); heterobifunctionalcross-linkers (e.g., amine reactive and sulfhydryl reactiveN-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), andcarbonyl-reactive and sulfhydryl-reactive 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH); and, as well, trifunctional cross-linkers (e.g.,4-azido-2-nitrophenylbiocytin-4-nitrophenyl ester). Particularlypreferred cross-linking agents are adipoyl chloride and1,4-dimaleimidobenzene.

Although the polymerized target may be provided essentially by itself atthe aqueous phase/liquid crystal interface, certain alternativeembodiments further comprise non-target molecules in combination withthe target molecules such that target and non-target molecules are inclose association, preferably cross-linked with each other. In someembodiments, the non-target molecule is a layer adsorbed or deposited atthe aqueous-liquid crystal interfaces to which a target molecule iscross-linked. In preferred embodiments, the non-target molecule is asurfactant, lipid or polymer that is adsorbed at the interface. In otherpreferred embodiments, the non-target molecule at the interface containsa amine or carboxylic acid group that is used to form a covalentcross-link with biomolecules at the interface.

Certain devices according to the invention further include an enhancingagent present in the aqueous phase that enhances the change inorientation of the liquid crystal upon interaction of the bioagent andtarget. Enhancing agents useful in this regard include, but are notlimited to dilauroyl phosphatidylcholine (DLPC), phospholipids (such asDPPC, DMPC), glycolipids, saturated and unsaturated lipids, fatty acids,polymers that cause homeotropic anchoring, anionic surfactants, cationicsurfactants, non-ionic surfactants, zwitterionic surfactants,amphiphilic molecules that generate known orientations of liquidcrystals. In general, enhancing agents that generate known orientationsof liquid crystal at the aqueous-liquid crystal interface are useful forthis purpose.

In one aspect of the invention, a method for preparing a liquid crystaldevice for detecting the presence of a bioagent in a sample is provided.Such methods include steps of: (a) dissolving a cross-linking agent intoa liquid crystal; and (b) adding a target of a bioagent to an aqueousphase that contacts said liquid crystal to cause an interfacial reactionbetween the target and the cross-linking agent. The interaction providesa membrane containing a polymerized target at the interface of theliquid crystal, the polymerized target positioned to interact with abioagent contacted with the membrane. Subsequent interaction betweenbioagent and target causes an orientation change in the liquid crystalthereby indicating the presence of the bioagent.

In yet another aspect of the invention, methods for detecting thepresence of a bioagent in a sample are provided. Such methods includesteps of: (a) forming a membrane containing a polymerized target for anenzyme at an interface between a liquid crystal and an aqueous phase;and (b) introducing a bioagent into the aqueous phase whereininteraction of the bioagent and polymerized target leads to a change inthe orientational order of the liquid crystal. The orientational changeindicates the presence of the bioagent in the sample.

In certain embodiments, the bioagent is an enzyme and the target is asubstrate for the enzyme. Preferred embodiments are directed to thedetection of proteases, metalloproteases and, most preferably,neurotoxins with metalloprotease activities. Accordingly, the substratein such methods is a peptide cleavable by the neurotoxin. For example,certain methods detect the presence of botulinum toxin using assubstrate a peptide cleavable by botulinum toxin (e.g., SNAP-25 or acleavable fragment thereof). In other embodiments, the enzyme detectedis a neurotoxin with phospholipase A₂ activity and the substrate is a1,2-diacyl-3-sn-phosphoglyceride containing an sn-2 ester bond.

In yet other embodiments, the method according to the invention detectsthe presence of an antibody in a sample by using as target an antigenrecognized by the respective antibody. As well, a sample may contain anantigen as a bioagent with the target then being an antibody thatrecognizes the antigen.

Certain methods according to the invention further include an enhancingagent present in the aqueous phase that enhances the change inorientation of the liquid crystal upon interaction of the bioagent andtarget. Enhancing agents useful in this regard include, but are notlimited to, L-α-dilauroyl phophatidylcholine (L-DLPC), phospholipids(such as DPPC, DMPC), glycolipids, saturated and unsaturated lipids,fatty acids, polymers that cause homeotropic anchoring, anionicsurfactants, cationic surfactants, non-ionic surfactants, zwitterionicsurfactants, amphiphilic molecules that generate known orientations ofliquid crystals. In general, enhancing agents that generate knownorientations of liquid crystal at the aqueous-liquid crystal interfaceare useful for this purpose.

It can be appreciated that devices and methods according to theinvention permit the incorporation of widely differing target forbioagents, including but not limited to proteins and lipids. It can alsobe appreciated that a variety of different interactions between bioagentand target can be detected by the invention, including, e.g.,disruption, perturbation and/or degradation of target by bioagent. Aswell, fabrication methods according to the invention provide substratecontaining membranes that can be tuned in thickness to balance the needfor sensitivity and robustness.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the enzymatic cleavage of polymermembrane film at the aqueous-5CB interface and the subsequent lipidmonolayer formation.

FIG. 2. Schematic representation of the polymerization of peptide at theaqueous-5CB interface and structures of 5CB and the peptide used in thisexperiment.

FIG. 3. Optical textures of 5CB mixed with adipoyl chloride (1.3 wt. %)after (a) 0 min and (b) 2 h contact with aqueous peptide solution (0.020mM, pH 11.0), and then (c) 30 min after contact with a dispersion ofvesicles of L-DLPC in water (0.1 mM, pH 8.0).

FIG. 4. Optical textures of 5CB fabricated with polymer membrane after(a) 0 min and (b) 10 min contact with a dispersion of vesicles of L-DLPC(0.1 mM) in water (pH 8.0). The sample in (a) and (b) was incubated inaqueous trypsin solution (21 μM, pH 8.0) for 2 h at rt before theaddition of L-DLPC; Optical textures of 5CB fabricated with polymermembrane after (a) 0 min and (b) 10 min contact with a dispersion ofvesicles of L-DLPC (0.1 mM) in water (pH 8.0). The sample in (c) and (d)was incubated in aqueous BSA solution (21 μM, pH 8.0) for 2 h at rtbefore the addition of L-DLPC.

FIG. 5. Optical images (transmission through crossed polars) of liquidcrystals in contact with polymerized membrane after incubation in (a) 2nM, (b) 20 nM, (c) 0.2 μM, and (d) 21 μM trypsin in water (pH 8) for 2h, and then after 30 min of contact with 0.1 mM L-DLPC in water (pH 8).

FIG. 6. Fabrication of enzymatically degradable polymer membrane atinterface between liquid crystal and aqueous phase.

FIG. 7. Degradation of polymer membrane and the subsequent assembly ofphospholipids at the interface between liquid crystals and aqueousphases.

FIG. 8. Optical images (transmission through crossed polars) of liquidcrystals after incubated in (a) 21 μM trypsin or (b) 21 μM BSA in water(pH 8) for 2 h, and then in 0.1 mM L-DLPC in water (pH 8).

FIG. 9. Optical images (transmission through crossed polars) of liquidcrystals after incubation in (a) 2 nM, (b) 20 nM, (c) 0.2 μM, and (d) 21μM trypsin in water (pH 8) for 2 h, and then 0.1 mM L-DLPC in water (pH8).

FIG. 10. Optical images (transmission through crossed polars) of liquidcrystals in contact with polymerized membrane of substrate for BoNT/Aafter incubation in (a) water (pH 8) or (b) 50 nM BoNT/A light chain inwater (pH 8) for 24 h, and then in 0.1 mM L-DLPC in water (pH 8).

FIG. 11. Schematic illustrate of the use of dimaleimide cross-linkersand HS-containing peptides to prepare a polymerized peptide membrane.

FIG. 12 Optical images (crossed polars) of (a) pure 5CB and (b) 5CB incontact with a polymer membrane fabricated according to the chemistryshown in FIG. 1, after 0 min and 1 h contact with the vesicle dispersionof L-DLPC in water (0.1 mM; pH 8.0).

FIG. 13. Immobilization of a 17-amino acid oligopeptide at a mixedmonolayer presenting carboxylic acid groups and tetra(ethylene glycol)groups through activation of the carboxylic acid groups by EDC/NHS.

FIG. 14. PM-IRRAS spectra of a SAM derived from EG6-CO₂H thiol on gold(a) before and (b) after 1.5 h of reaction with EDC/NHS, and then (c)after 1.5 h of reaction with 17-amino acid oligopeptide.

FIG. 15. Polarized light micrographs of 5CB (crossed polars) (a) incontact with PBS buffer (pH 7.6), (b) after 1 h of contact with anaqueous mixture of carboxyl-/hydroxyl-terminated surfactants (0.80/0.20mM; PBS buffer, pH 7.6), (c) after 1.5 h of reaction with EDC/NHS(50/200 mM; PBS buffer, pH 7.6), and then (d) after 1.5 h of contactwith an aqueous solution of 17-amino acid oligopeptide (0.020 mM; PBSbuffer, pH 7.6).

FIG. 16. Polarized light micrographs of (a) 5CB (crossed polars) ladenwith a mixture of carboxyl-/hydroxyl-terminated surfactants after 1.5 hcontact with 17-amino acid oligopeptide. Polarized light micrographs of5CB (crossed polars) laden with the NHS-activated mixed monolayer after1.5 h of contact with (b) ethanolamine, (c) lysine, and (d) polylysinesolution (PBS buffer, pH 7.6).

FIG. 17. Polarized light micrographs (crossed polars) of 5CB laden withthe mixed monolayer modified with 17-amino acid oligopeptide aftercontacting with trypsin, a mixture of trypsin and trypsin inhibitor, orBSA solution in 30 mM HEPES buffer containing 20 mM CaCl₂ (pH 8.0) forthe indicated time periods.

DETAILED DESCRIPTION OF THE INVENTION I. In General

Before the present materials and methods are described, it is understoodthat this invention is not limited to the particular methodology,protocols, materials, and reagents described, as these may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. As well, the terms “a” (or “an”),“one or more” and “at least one” can be used interchangeably herein. Itis also to be noted that the terms “comprising”, “including”, and“having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the materials, chemicals,instruments, statistical analysis and methodologies which are reportedin the publications which might be used in connection with theinvention. All references cited in this specification are to be taken asindicative of the level of skill in the art. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

II. The Invention

In a first aspect, the present invention provides a liquid crystaldevice for detecting the presence of a bioagent in a sample. Such adevice includes: (a) a liquid crystal; (b) an aqueous phase positionedsuch that an interface exists between the liquid crystal and the aqueousphase; and (c) a membrane containing a polymerized target of a bioagent.The membrane is located at the interface between the liquid crystal andthe aqueous phase. Interaction of the bioagent with the polymerizedtarget causes an orientation change in the liquid crystal therebyindicating the presence of the bioagent in the sample.

As used herein, the term “bioagent” shall encompass a wide variety ofmolecules and assemblies of molecules such as viruses, bacteria,eukaryotic and prokaryotic cells to be detected in a fluid sample. Suchmolecules may be synthetic or natural in origin and include, but are notlimited to, protein, carbohydrate, lipid, nucleic acid, andorganic/inorganic small molecule entities. These molecules andassemblies may be complexes with metal ions. In certain embodiments, thebioagent is an enzyme and the target is a substrate for the bioagent.Preferred embodiments are directed to the detection proteases andmetalloproteases, including neurotoxins with metalloprotease activities.Accordingly, the substrate is a peptide cleavable by said neurotoxin.For example, certain devices detect the presence of botulinum toxinusing as substrate a peptide cleavable by botulinum toxin (e.g., SNAP-25or a cleavable fragment thereof). In other embodiments, the enzymedetected is a neurotoxin with phospholipase A₂ activity and thesubstrate is a 1,2-diacyl-3-sn-phosphoglyceride containing an sn-2 esterbond. In yet other embodiments, the liquid crystal device detects thepresence of an antibody in a sample by using an antigen recognized bythe respective antibody.

As used herein, the term “polymerized target” refers to biomolecules(e.g., proteins, peptides, oligopeptides, lipids, peptide amphiphiles,carbohydrates, nucleic acids, or small organic/inorganic molecules,molecules that interact with biological entities including prokaryoticand eukaryotic organisms) that have been cross-linked to forminterconnected molecular networks while still retaining the ability tointeract with a bioagent. The term “interact” in the context of targetand bioagent includes, but is not limited to, the disruption,perturbation and/or degradation of the target by the direct action ofthe bioagent. As described herein, polymerized targets are preferablyformed by cross-linking target molecules with the aid of a cross-linkingagent. The term “cross-linking agent” or “conjugation agent” shall referto the broad family of conjugation reagents known in the art to beuseful in conjugating (cross-linking) biomolecules. Cross-linking agentsuseful in the invention, particularly in the protein context, include,but are not limited to: zero length cross-linkers (e.g.,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC));homobifunctional cross-linkers (e.g., the NHS esterdithiobis(succinimidylpropionate (DSP), the imidoester dimethyladipimidate (DMA), sulfhydryl-reactive1,4-Di-[3′-(2′-pyridyldithio)priopionamido]butane (DPDPB), thedifluorobenzene derivative 1,5-difluoro-2,4-dinitrobenzene (DFDNB),formaldehyde, bis-epoxides, and hydrazides); heterobifunctionalcross-linkers (e.g., amine reactive and sulfhydryl reactiveN-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), andcarbonyl-reactive and sulfhydryl-reactive 4-(4-N-maleimidophenyl)butyricacid hydrazide (MPBH); and, as well, trifunctional cross-linkers (e.g.,4-azido-2-nitrophenylbiocytin-4-nitrophenyl ester). Particularlypreferred cross-linking agents are adipoyl chloride and1,4-dimaleimidobenzene. Conjugation of proteins to lipids may beaccomplished, e.g., via the NHS ester of a fatty acid, carbodiimidecoupling, glutaraledyde coupling, DMS cross-linking, N-succinimidyl3-(2-pyridyldithio)propionate (SPDP)-modified lipid derivatives, orsuccinimidyl-4-(para-maleimidophenyl)butyrate (SMPB)-modified lipidderivatives. In the antibody context, cross-linking techniques are knownin the art and exemplary such techniques include NHSester-maleimide-mediated conjugation, glutaraldehyde-mediatedconjugation, reductive-amination-mediated conjugation, and disulfideexchange reactions. An extensive list of suitable cross-linking agentsis presented in “Bioconjugate Techniques” by Greg T. Hermanson, AcademicPress, 1996, ISBN0-12-342336-8, incorporated herein by reference.

Although the polymerized target may be provided essentially alone at theaqueous phase/liquid crystal interface, certain alternative embodimentsfurther comprise non-target molecules in combination with the targetmolecules such that target and non-target molecules are in closeassociation, preferably cross-linked with each other. “Non-target”molecules are provided in combination with target molecules but are notdirectly disrupted, perturbed, and/or degraded by the bioagent.Non-target molecules may be indirectly acted upon by bioagents becausethey are coupled to the target and, consequently, effect underlyingliquid crystal orientation. In one embodiment, the non-target moleculeis a spontaneously adsorbed or deposited monolayer to which a targetmolecule is cross-linked. Preferred classes of non-target molecule arepolymers, surfactants and lipids that are assembled at the liquidcrystal-aqueous interface. Non-target molecules may be provided asmixtures or combinations. A particularly preferred class of non-targetmolecules are lipids and surfactants that contain non-ionic groups suchas ethylene glycol. A preferred embodiment of the invention involves theuse of surfactants that incorporate ethylene glycol head groups such astetraethylene glycol monotetradecyl ether (C14EG4) and3,6,9,12,15-pentaoxanonacosanoic acid (C14EG4-CO₂H), and mixtures ofthese two compounds. A further preferred embodiment of the inventionuses NHS/EDC to activate the carboxylic acid group of3,6,9,12,15-pentaoxanonacosanoic acid to form a cross-linked network ofthe target and non-target molecules at the interface between the liquidcrystal and aqueous phase.

Furthermore, different types of liquid crystals can be employed in thepresent invention, including nematic and smectic liquid crystals, andthermotropic liquid crystals, as will be evident to those skilled in theart of liquid crystals. Examples of suitable liquid crystals, include,but are not limited to, 4-cyano-4′-pentylbiphenyl (5CB), 7CB, and 8CB. Alarge listing of suitable liquid crystals is presented in “Handbook ofLiquid Crystal Research” by Peter J. Collings and Jay S. Patel, OxfordUniversity Press, 1997, ISBN 0-19-508442-X, incorporated herein byreference. Polymeric liquid crystals are also suitable for use in thedevice and methods of the present invention. Other liquid crystals arenematic liquid crystals such as E7, smectic liquid crystals,thermotropic liquid crystals, lyotropic liquid crystals, polymericliquid crystals, cholesteric liquid crystals and ferroelectric liquidcrystals. In a preferred embodiment of the present invention, the liquidcrystal deposited in the device is 4-cyano-4′-pentylbiphenyl (5CB).Although various types of liquid crystal may be employed, nematic andthermotropic liquid crystals are preferred. However, smectic liquidcrystals formed from 8CB are also suitable for use in the presentinvention. Suitable liquid crystals further include smectic C, smecticC*, blue phases, cholesteric phases, smectic A, and polymeric liquidcrystals. It is further envisioned that LCs useful in the invention mayfurther include additions of dopants such as, but not limited to, chiraldopants as described by Shitara H, et al. (Chemistry Letters 3: 261-262(1998)) and Pape, M., et al. (Molecular Crystals and Liquid Crystals307: 155-173 (1997)). The introduction of a dopant permits manipulationof the liquid crystal's characteristics including, but not limited to,the torque transmitted by the liquid crystal to an underlying surface.Other dopants, such as salts, permit manipulation of the electricaldouble layers that form at the interfaces of the liquid crystals andthus permit manipulation of the strength of anchoring of the liquidcrystal at the interface. A number of methods for preparing interfacesbetween liquid crystals and aqueous phases lie within the scope of thepresent invention. An approximately planar interface can be prepared bya film of liquid crystal in contact with an aqueous phase, oralternatively a curved interface can be prepared by using a droplet ofliquid crystal dispersed in an aqueous phase. The scope of the inventionis not limited by the methods by which interfaces between aqueous phasesand liquid crystals can be prepared by those skilled in the art.

Certain devices according to the invention further include an enhancingagent present in the aqueous phase that enhances the change inorientation of the liquid crystal upon interaction of the bioagent andtarget. Enhancing agents useful in this regard include, but are notlimited to, L-α-dilauroyl phophatidylcholine (L-DLPC), phospholipids(such as DPPC, DMPC), glycolipids, saturated and unsaturated lipids,fatty acids, polymers that cause homeotropic anchoring, anionicsurfactants, cationic surfactants, non-ionic surfactants, zwitterionicsurfactants, amphiphilic molecules that generate known orientations ofliquid crystals. In general, enhancing agents that generate knownorientations of liquid crystal at the aqueous-liquid crystal interfaceare useful for this purpose.

In another aspect of the invention, a method for preparing a liquidcrystal device for detecting the presence of a bioagent in a sample isprovided. Such methods include steps of: (a) dissolving a cross-linkingagent into a liquid crystal, and (b) adding a target of a bioagent to anaqueous phase that contacts said liquid crystal to cause an interfacialreaction between the target and the cross-linking agent. The interactionprovides a membrane containing a polymerized target at the interface ofthe liquid crystal, the polymerized target positioned to interact with abioagent contacted with the membrane. Subsequent interaction betweenbioagent and target causes an orientation change in the liquid crystalthereby indicating the presence of the bioagent. As described above, themembrane may further comprise non-target molecules including, but notlimited to, self-assembled monolayers (SAMs) with which target moleculesare cross-linked.

In yet another aspect of the invention, methods for detecting thepresence of a bioagent in a sample are provided. Such methods includesteps of: (a) forming a membrane containing a polymerized target for anenzyme at an interface between a liquid crystal and an aqueous phase;and (b) introducing a bioagent into the aqueous phase whereininteraction of the bioagent and polymerized target leads to a change inthe orientational order of the liquid crystal. The orientational changeindicates the presence of the bioagent in the sample.

In certain embodiments, the bioagent is an enzyme and the target is asubstrate for the bioagent. Preferred embodiments are directed to thedetection of neurotoxins with metalloprotease activities. Accordingly,the substrate is a peptide cleavable by said neurotoxin. For example,certain methods detect the presence of botulinum toxin using assubstrate a peptide cleavable by botulinum toxin (e.g., SNAP-25 or acleavable fragment thereof). The 206 amino acid sequence for humanSNAP-25 (SEQ ID NO:1) is publicly available via the National Center forBiotechnology Information (NCBI) under accession no. P60880. The aminoacid sequence for human SNAP-25 and related information provided ataccession no. P60880 as of this application's filing date areincorporated herein by reference in their entirety. In otherembodiments, the enzyme detected is a neurotoxin with phospholipase A₂activity and the substrate is a 1,2-diacyl-3-sn-phosphoglyceridecontaining an sn-2 ester bond. A wide variety of such enzymes, namelyneurotoxins, are known in the art and the present invention isparticularly advantageous for use in their detection. An extensive listof neurotoxins are described in Schiavo et al., “Neurotoxins affectingNeuroexocytosis,” Physiological Reviews 80:717-766 (2000), incorporatedherein by reference.

In yet other embodiments, the devices and methods include a membranethat incorporates a target molecule that is a binding group for bindinga protein of interest to the membrane. Accordingly, the inventionprovides in specific embodiments devices and methods for detecting thepresence of an antibody in a sample by using an antigen recognized bythe respective antibody. Alternatively, the invention provides specificembodiments in which the presence of an antigen in a sample is detectedby forming a membrane containing an antibody. In these preferredembodiments, the membrane containing the antibody (for antigendetection) or antigen (for antibody detection) is prepared usingsubstantially the same strategy as described above. In one preferredembodiment, the cross-linked membrane containing a protein or peptideantigen (for antibody detection) is prepared by adding adipoylchlorideto the liquid crystal and the antigen to the aqueous phase. The reactionbetween the antigen and adipoylchloride at the interface between theaqueous phase and liquid crystal leads to the formation of a polymerizedmembrane containing antigen. To detect the presence of antibody in asample, the sample containing antibody is added to the aqueous phase incontact with the polymerized membrane containing antigen. The binding ofthe antibody to the antigen containing membrane is detected by a changein order of the liquid crystal in contact with the membrane.

In preferred embodiment, enhancers are added to the aqueous phase toincrease the change in order of the liquid crystal when the antibody isbound. Enhancing agents useful in this regard include, but are notlimited to, L-α-dilauroyl phophatidylcholine (L-DLPC), phospholipids(such as DPPC, DMPC), glycolipids, saturated and unsaturated lipids,fatty acids, polymers that cause homeotropic anchoring, anionicsurfactants, cationic surfactants, non-ionic surfactants, zwitterionicsurfactants, amphiphilic molecules that generate known orientations ofliquid crystals. In general, enhancing agents that generate knownorientations of liquid crystal at the aqueous-liquid crystal interfaceare useful for this purpose. In other embodiments, the membrane isprepared as described above to contain the antibody; an antigencontained in a sample may then be detected via interaction betweenpolymerized antibody and respective antigen.

As noted above, preferred embodiments of the invention are directed to amethod that provides for the selective detection of an enzyme based onenzymatically cleavable peptide-containing membranes and liquidcrystals. The membrane including the polymerized substrate for aspecific enzyme or class of enzymes can be selectively degraded bycontact with the enzyme. The degradation of the polymeric membranefabricated at the interface between liquid crystals and aqueous phaseschanges the orientation of the liquid crystal in contact with theaqueous phase (FIGS. 1 a and 1 b). As a result, the presence of aprotein can be recognized by monitoring the change in orientation of aliquid crystal using optical or electrical methods well known to thoseskilled in the art. In some embodiments of the invention, the change inordering of the liquid crystal can be controlled by the addition of aphospholipid when exposed to aqueous phase (FIG. 1 c). A merit of thisdetection method is that the presence of an enzyme can be monitoredwithout a label attached to the analyte. The method also does notrequire a complicated surface patterning/functionalization procedure andspecial instrumentation. Therefore, in certain embodiments, the presentinvention offers efficient and cost-effective detection of biomolecules.

An illustrative example of a liquid crystal device, method offabrication, and use according to the invention will now be described.In this example, an initial step is the preparation of a polymermembrane containing peptide substrates that can be recognized by anenzyme. FIG. 2 illustrates the polymerization of a peptide betweenthermotropic liquid crystals and aqueous phases. A natural peptidesubstrate was used in this example to show the possibility of practicalapplications of the invention. The 17-amino acid peptide shown in FIG. 2(SEQ ID NO:3) contains residues 187-203 of SNAP-25, a 206-residueprotein. The 17-mer peptide substrate is known to be effectively cleavedby botulinum neurotoxin type A (BoNT/A) metalloprotease, which is themost poisonous substance known. The methionine (M) at position 202 wasreplaced with the isosteric amino acid norleucine (Nle) to preventoxidation of the peptide. The 17-amino acid peptide was synthesized andthe crude peptide was purified by chromatography over a preparativescale C-18 column. The reverse-phase high performance liquidchromatography (HPLC) analyses confirmed the peptide had >98% purity,and MALDI-TOF mass spectrometry spectra confirmed its predicted mass.

The 17-amino acid peptide was polymerized with adipoyl chloride as acrosslinking agent at the interface between thermotropic liquid crystalsand aqueous phases. The reaction of the peptide with adipoyl chlorideleads to the formation of amide interpeptide linkages through lysines(K) in its sequence. It has been previously reported that abiodegradable polymer can be synthesized by the reaction of amino acidswith adipoyl chloride. The adipoyl chloride was added to the liquidcrystal 4-cyano-4′-pentylbiphenyl (5CB, shown in FIG. 2) andhomogeneously mixed by using a vortex mixer. The composition of themixture was prepared to be 1.3 wt. % adipoyl chloride in 5CB. Themixture was deposited into the pores of gold TEM grids supported onoctyltrichlorosilane (OTS)-coated glass slides. Then, the peptide inaqueous NaOH solution (0.20 mM, pH 11.0) was introduced to the sample(FIG. 2). After being incubated for 2 hours at room temperature, thepeptide solution was removed using a pipette and the sample was rinsedwith water more than five times. In other embodiments of the inventiondroplets of liquid crystal containing cross-linking agent can bedispersed into an aqueous solution containing the substrate to bepolymerized at the interface between the liquid crystal and aqueousphase.

FIG. 3 shows the optical textures of 5CB monitored duringpolymerization. All optical textures reported herein were measuredbetween crossed polarizers. The optical texture of 5CB doped withadipoyl chloride became bright immediately after contacting with theaqueous peptide solution (FIG. 3 a). Since it is known that theOTS-coated glass slides cause homeotropic anchoring of 5CB on thesurface, the orientation of 5CB at the aqueous-5CB interface istherefore near planar or planar. Under these circumstances, the tiltangle of 5CB relative to the surface normal is gradually decreases from˜0° at the OTS-treated glass to ˜90° at the aqueous-5CB interface. Whenthe tilt angle of the liquid crystal continuously varies across thefilm, nematic 5CB, because it possesses two direction-dependentrefractive indices (is birefringent), can rotate the plane ofpolarization of the light, thus leading providing a bright opticalappearance. FIG. 3 b shows the optical image of 5CB doped with adipoylchloride after 2 h of contact with the aqueous peptide solution at roomtemperature. The optical image became darker and darker during theformation of polymer at the aquesous-5CB interface.

To confirm the formation of polymerized peptide substrate, the aqueouspeptide solution was removed using a pipette and a dispersion ofvesicles of L-α-dilauroyl phosphatidylcholine (L-DLPC) in water (0.1 mM,pH 8.0) was subsequently introduced into the sample after washing withwater several times and storing in water for 2 h at rt. In the absenceof the polymer membrane, contact of an aqueous dispersion of L-DLPC withthe interface of 5CB is known to cause the spontaneous transfer of thelipids onto the aqueous-5CB interface leading to a homeotropic alignmentof 5CB at the interface. The homeotropic alignment can be confirmed byblack optical appearance of 5CB between crossed polars and a conoscopicimage. The inventors determined if the polymer membrane fabricated atthe aqueous-5CB interface would block the transfer of phospholipids ontothe interface and thus maintain the orientation of 5CB near planar orplanar at the interface. After 30 min of immersion, the optical texturedid not show the formation of black domains in the liquid crystal,confirming that the polymer membrane at the aqueous-5CB interfaceblocked the transfer of lipids onto the interface (FIG. 3 c).

The enzyme trypsin was used to test the cleavage of polymer membrane.Trypsin is known to be selective in its cleavage of peptide bonds.Trypsin cleaves peptide bonds after (on the C-terminal side of) lysine(K) and arginine (R) if the next residue is not proline (P). The 17-merpeptide (FIG. 2) used in this example, therefore, contains four cleavagesites by trypsin after two lysine and two arginine residues in itssequence. To test the cleavage of polymer membrane at the aqueous-5CBinterface, an aqueous trypsin solution (˜21 μM, pH 8.0) was introducedinto an aqueous solution in contact with the polymerized membrane. ThepH of the trypsin solution was adjusted to 8 by a dilute aqueous NaOHsolution to maximize the reactivity of trypsin. After being incubatedfor 2 hours at room temperature, the trypsin solution was removed by apipette and the sample was rinsed with slightly basic water (pH 8)several times. To confirm the degradation of the polymer membrane bytrypsin, an aqueous solution containing a dispersion of vesicles ofL-DLPC (0.10 mM, pH 8.0) was introduced into the sample. It wasanticipated that the degradation of the polymer membrane by trypsinwould allow the transfer of phopholipids onto the aqueous-5CB interface(FIG. 1 c), thus leading to an orientational transition in the liquidcrystal that could be monitored optically.

The initial bright optical appearance of 5CB between crossed polarsindicates that the orientation of 5CB is near planar at the aqueous-5CBinterface (FIG. 4 a). In contrast, after 10 min of contact with anaqueous dispersion of vesicles of L-DLPC (0.10 mM, pH 8.0), the opticaltexture of 5CB became black (FIG. 4 b). The black appearance correspondsto liquid crystal anchored perpendicular to both the aqueous-5CB and the5CB-OTS interfaces (FIG. 1 c). In the case that the orientation of 5CBis perpendicular to both interfaces, the liquid crystal does not rotatethe polarization plane of light leading to its dark appearance betweencrossed polars. The homeotropic anchoring was also confirmed by theconoscopic image (black cross in the inset of FIG. 4 b).

To confirm that the orientational transition arises from the degradationof polymer membrane by the enzyme trypsin, a protein was tested whichcan not hydrolyze the polymer membrane. Instead of the trypsin solution,an aqueous solution of bovine serum albumin (BSA) (21 μM, pH 8.0) wasincubated against the polymerized peptide membrane fabricated at theaqueous-5CB interface.

After being incubated for 2 h at room temperature, the BSA solution wasremoved and the sample was rinsed with deionized water more than fivetimes. Then, like the previous experiment with trypsin, an aqueousdispersion of vesicles of L-DLPC (0.10 mM, pH 8.0) was introduced intothe sample. In contrast to the results obtained with trypsin, theoptical appearance of 5CB remained bright even after 10 min contact withthe L-DLPC dispersion (FIGS. 4 c and 4 d). The homeotropic anchoring of5CB was not observed even after 1 h of contact with L-DLPC. The resultsdemonstrate that the orientational transition of 5CB observed with thesample treated with trypsin (FIGS. 4 a and 4 b) arises from theselective cleavage of the polymer membrane by trypsin. The methodpresented here, therefore, can be used to differentiate proteins thatcan/cannot cleave polymer membrane when an appropriate peptide substrateis employed.

Aqueous trypsin solutions of various concentrations ranging from 2 nM to21 μM (pH 8.0) were prepared and tested using a polymer membraneprepared as described above. After 30 min of immersion in aqueousdispersions of vesicles of L-DLPC (0.10 mM, pH 8.0), the sampleincubated in 2 nM trypsin solution showed small black domains (FIG. 5a). In contrast, the samples incubated in trypsin solutions of higherconcentrations (20 nM, 0.2 μM, and 21 μM) exhibited transition fromplanar to homeotropic anchoring in most areas (FIGS. 5 b, 5 c, and 5 d)indicating degradation of polymer membrane by trypsin. These resultsdemonstrate detection of trypsin at concentrations near 2 nM under theabove described conditions. It is anticipated that the detection limitcan be lowered by optimizing experimental conditions such as thethickness of polymer membrane and temperature. The thickness of polymerfilm can be decreased by decreasing the concentrations of adipoylchloride in 5CB and peptide in water.

A convenient method for detection of an enzyme using an enzymaticallycleavable protein-containing membrane at the interface between liquidcrystal and aqueous phases is demonstrated herein. The method can beused to monitor the presence of a variety of enzymes when an appropriatepeptide substrate for a specific enzyme is incorporated. It should benoted that the 17-amino acid peptide used in the foregoing example isthe substrate that is effectively cleaved by botulinum neurotoxin type A(BoNT/A) which is an extremely lethal substance. Thus, the methoddisclosed herein can be directly applied to detect the BoNT/A withoutany major modification. Fast detection of BoNT/A is important for publichealth and food safety. The above example using an oligomeric peptidesubstrate is illustrative, and the method can also be applied to thepreparation of peptide substrates that are polymers, including proteins.As well, the method is not limited to the use of adipoylchloride as thecross linker—many cross-linking agents for peptides, proteins, nucleicacids are known to those skilled in the art. Various cross-linkingagents containing two maleimide groups and peptides or proteinscontaining —SH groups are shown in FIG. 11.

Accordingly, it is contemplated that the polymeric material used tofabricate the polymerized substrates incorporate peptide sequences thatserve as recognition elements for the analyte/bioagent to be detected.It is noted that the peptide sequences can serve as recognition elementsfor other types of agents, such as chemical agents, without deviatingfrom the scope of the present invention. The peptide sequences provide amolecular basis for sensor specificity, as well as, the mechanism bywhich the polymerized substrate erodes (i.e., peptide bond cleavage)when exposed to the predetermined bioagent. It can be appreciated that apolymerized substrate acting as biological sensor possesses severaladvantages within a microfluidic platform. For example, small amounts ofreagents are needed to produce these polymerized substrates. Further,these substrates are thin, lowering the diffusion path length of theagent to be detected (i.e. large enzymes, toxins and proteases).

In yet other embodiments, the method detects the presence of an antibodyin a sample by using an antigen recognized by the respective antibody.The invention provides specific embodiments in which the presence of anantigen in a sample is detected by forming a membrane containing anantibody. In these preferred embodiments, the membrane containing theantibody (for antigen detection) or antigen (for antibody detection) isprepared using strategies substantially similar to those describedabove. In one preferred embodiment, the cross-linked membrane containinga protein or peptide antigen (for antibody detection) is prepared byadding adipoylchloride to the liquid crystal and the antigen to theaqueous phase. The reaction between the antigen and adipoylchloride atthe interface between the aqueous phase and liquid crystal leads to theformation of a polymerized membrane containing antigen. To detect thepresence of antibody in a sample, the sample containing antibody isadded to the aqueous phase in contact with the polymerized membranecontaining antigen. The binding of the antibody to the antigencontaining membrane is detected by a change in order of the liquidcrystal in contact with the membrane.

In preferred embodiment, enhancers are added to the aqueous phase toincrease the change in order of the liquid crystal when the antibody isbound. Enhancing agents useful in this regard include, but are notlimited to, L-α-dilauroyl phophatidylcholine (L-DLPC), phospholipids(such as DPPC, DMPC), glycolipids, saturated and unsaturated lipids,fatty acids, polymers that cause homeotropic anchoring, anionicsurfactants, cationic surfactants, non-ionic surfactants, zwitterionicsurfactants, amphiphilic molecules that generate known orientations ofliquid crystals. In general, enhancing agents that generate knownorientations of liquid crystal at the aqueous-liquid crystal interfaceare useful for this purpose. In other embodiments, the membrane isprepared as described above to contain the antibody; an antigencontained in a sample may then be detected via interaction betweenpolymerized antibody and respective antigen.

The following examples are offered for further illustrative purposesrelated to the above-described devices and methods, and are not intendedto limit the scope of the present invention in any way. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and the following examples and fall within thescope of the appended claims.

III. Examples Example 1 Preparation of a Device Containing a PolymerizedMembrane at the Interface between a Nematic Liquid Crystal and anAqueous Phase—the Polymerized Membrane Containing the Peptide Substratefor Botulinum Toxin

This example describes the polymerization of peptides with adipoylchloride at interfaces between thermotropic liquid crystals and aqueousphases. The sequences of peptides used in this example are shown below.The HPLC analyses confirmed the peptides had >98% purity, and MALDI-MSspectra confirmed their structures.

Peptide I: NH₂-CSNKTRIDEANQRATK{Nle}LC-amide (SEQ ID NO: 2) Peptide II:NH₂-SNKTRIDEANQRATK{Nle}L-amide (SEQ ID NO: 3)

These peptides were polymerized with adipoyl chloride as a cross linkerat the interface between liquid crystals and aqueous phases. The adipoylchloride was added to the liquid crystal 4-cyano-4′-pentylbiphenyl (5CB)and homogeneously mixed by sonication. The composition of the mixturewas about 1.5 wt. % adipoyl chloride in 5CB. The mixture was depositedinto the pores of gold TEM grids supported on octyltrichlorosilane(OTS)-coated glass slides. The peptide in aqueous NaOH solution (0.20mM, pH 11) was introduced to the sample (FIG. 6). After polymerizationfor 2 hours at room temperature, the peptide solution was removed andthe sample was rinsed with a slightly basic water (pH 8) more than fivetimes.

Example 2 Degradation of a Substrate Containing Polymerized MembraneUsing Trypsin

In this example, trypsin was selected as a model enzyme to test thecleavage of a polymer membrane. Trypsin is known to be selective in itscleavage of peptide bonds. Trypsin only cleaves the peptide bonds after(on the C-terminal side of) lysine (K) and arginine (R) if the nextresidue is not proline (P). Therefore, the peptide I and II contain fourcleavage sites by trypsin after two lysine and two arginine residues inits sequence. An aqueous trypsin solution (˜21 μM, pH 8) was introducedinto the sample. After being incubated for 2 hours at room temperature,the trypsin solution was removed and the sample was rinsed with slightlybasic water (pH 8) several times. To confirm the degradation of thepolymer membrane by trypsin, an aqueous solution containing a dispersionof vesicles of L-α-dilauroyl phophatidylcholine (L-DLPC) was introducedinto the sample. The degradation of the polymer membrane allows theassembly of phopholipids at the interface between liquid crystals andaqueous phases (FIG. 7). As a result, the degradation of the polymermembrane can be confirmed by monitoring orientational transitions inliquid crystals triggered by the spontaneous assembly of thephospholipid.

Within 10 min of immersion in an aqueous solution containing adispersion of vesicles of L-DLPC, the optical texture viewed betweencrossed polars became black (FIG. 8 a). The black domains correspond toliquid crystal that is anchored perpendicular to both the liquidcrystal-water and the liquid crystal-OTS interfaces (FIG. 7). As acontrol, a protein which can not cleave the polymer membrane was alsotested. An aqueous solution of bovine serum albumin (BSA) (21 μM, pH 8)was introduced into the polymer sample instead of the trypsin solution.After reacting for 2 hours at room temperature, the BSA solution wasremoved and the sample was rinsed with slightly basic water more thanfive times. Even after an aqueous solution containing a dispersion ofvesicles of L-DLPC was introduced into the sample, the liquid crystalremained bright (FIG. 8 a-b). These results, as a whole, show that themethod allows differentiation of proteins that can/cannot cleave thepolymer membranes by using liquid crystals.

The inventors investigated aqueous trypsin solutions of variousconcentrations ranging from 2 nM to 21 μM (pH 8) for degradation of thepolymer membrane. After the introduction of L-DLPC, all samples showedregions of homeotropic alignment indicating the activity of the enzyme.The highest concentrations caused the liquid crystal to appear almostcompletely black between crossed polars (FIG. 9 a-d).

Example 3 Detection of the Light Chain of Botulinum Toxin

The peptides used in this example (Peptide I and II) contain residues187-203 of SNAP-25. The peptides are known to be effectively cleaved bybotulinum neurotoxin type A (BoNT/A), the most lethal substance known.Therefore, the system used for trypsin can be applied to detect BoNT/Awithout major modification. The procedure for polymerization was same asdescribed above (see Example 1). After polymerization, water (pH 8) oran aqueous solution of BoNT/A light chain (pH 8) was introduced to thesample. After incubated for 24 hours at room temperature, the solutionswere removed and the sample was rinsed with water more than 5 times.When a dispersion of vesicles of L-DLPC was introduced to the sample,only the sample incubated in aqueous BoNT/A light chain solutionexhibited the transition of 5CB anchoring from planar to homeotropic atthe aqueous-5CB interface (FIG. 10 a-b). The results indicates that thepolymer membrane is cleaved by BoNT/A light chain, and thus the systemcan be used to detect the presence of BoNT/A. This system provides anovel method for the detection of various biomolecules when anappropriate peptide substrate is incorporated.

Example 4 Formation of a Polymerized Peptide Membrane Using MaleimideCross-linkers

The inventors synthesized the 19-amino acid peptide (SEQ ID NO:2), whichcontains residues 187-203 of SNAP-25 and two cysteines at both ends. Thesulfhydryl groups of terminal cysteines provide the opportunity toconjugate the synthetic peptide to the maleimide containing crosslinker(FIG. 11).

The 1,4-dimaleimidobenzene (0.0035 g) was mixed with the liquid crystal4-cyano-4′-pentylbiphenyl (5CB, 0.2076 g) by using a vortex mixer. Themixture was deposited into the pores of gold TEM grids supported onoctyltrichlorosilane (OTS)-coated glass slides. The 0.20 mM solution ofthe 19-amino acid peptide in phosphate buffer (aqueous 20 mM; pH 7.0)was prepared and introduced to the sample. After being incubated for 3 hat room temperature, the peptide solution was removed using a pipetteand the sample was rinsed with water several times. The sample wasstored in an oven held at 36° C. for 2 h.

To confirm the formation of polymer at the aqueous-5CB interface, adispersion of vesicles of L-a-dilauroyl phosphatidylcholine (L-DLPC) inwater (0.1 mM; pH 8.0) was introduced to the sample. In the absence ofthe polymerized membrane contact of a vesicle dispersion of L-DLPC with5CB leads to a homeotropic alignment of 5CB at the interface withinabout 20 min (FIG. 12 a). In contrast, after even 1 h of immersion, theoptical texture of 5CB with the polymer membrane showed only small areasof homeotropic alignment of 5CB indicating the formation of a polymermembrane at the interface by using a dimaleimide-based cross-linker(FIG. 12 b).

Accordingly, various aspects of the present have been shown including:(a) the formation of devices that include a polymerized membrane betweena liquid crystal and an aqueous phase, including an example in which themembrane contains a substrate for an enzyme; (b) the practice of methodswherein the addition of a cross linking agent to the liquid crystal anda substrate for an enzyme to the aqueous phase leads to the formation ofa polymerized membrane containing the substrate at the interface betweenthe liquid crystal and the aqueous phase; and (c) the performance ofmethods wherein a polymerized membrane containing a substrate to trypsinand botulinum toxin prepared at the interface between the liquid crystaland aqueous phase is used to report the presence of trypsin and thelight chain of botulinum toxin.

Example 5 Amplification of Enzymatic Activities Using OligopeptidesCross-linked at Interfaces Between Aqueous Phases and Liquid Crystals

In this example, a strategy for cross-linking of oligopeptides ataqueous-liquid crystal interfaces is provided. This example alsodescribes the response of liquid crystals to the cross linking ofoligopeptides and the cleavage of the oligopeptides by an enzyme. Totest the strategy, the inventors used a 17-amino acid oligopeptide (FIG.13; SEQ ID NO:3) that is the substrate of botulinum neurotoxin type A(BoNT/A) because the ultimate goal of this example is the detection ofBoNT/A by using this oligopeptide-modified interface. In view of thedangers associated with botulinum toxin, the inventors used trypsin as amodel enzyme that also can cleave the 17-amino acid oligopeptide.

This example is organized into three parts. First, the preparation of amixed monolayer of OH- and CO₂H-terminated surfactants at aqueous-liquidcrystal interfaces and the immobilization of the oligopeptide at themixed monolayer through the N-hydroxylsuccinimide (NHS)-activation ofcarboxylic acid groups in the monolayer is described (FIG. 13). Second,the orientational response of liquid crystals to the immobilization ofthe oligopeptide is descrribed and a possible mechanism for theanchoring transition is proposed. Finally, the oligopeptide-modifiedinterface is used to detect an enzymatic activity and demonstrate theselectivity of this detection system.

Experimental Section

Materials. The 17-amino acid oligopeptide was synthesized at theBiotechnology Center at the University of Wisconsin-Madison using a Fmocprotocol with an Applied Biosystems Synergy 432A instrument. A detailedprocedure of the synthesis has been previously reported. All aqueoussolutions were prepared using deionized water. Deionization of adistilled water source was performed using a Milli-Q system (Millipore,Bedford, Mass.) to give water with a resistivity of 18.2 MΩ cm. Theliquid crystal 4-cyano-4′-pentylbiphenyl (5CB) was purchased from EMIndustries (Hawthorne, N.Y.). Tetraethylene glycol monotetradecyl ether(C14EG4), N-hydroxylsuccinimide (NHS), ethanolamine,octyltrichlorosilane (OTS), trypsin, trypsin-chymotrypsin inhibitor fromGlycine max (Bowman-Birk inhibitor), DL-lysine, and poly-L-lysinehydrobromide (M.W. 1,000-4,000) were purchased from Aldrich (Milwaukee,Wis.). 1-Ethyl-2-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)was purchased from Pierce Biotechnology (Rockford, Ill.).3,6,9,12,15-Pentaoxanonacosanoic acid (C14EG4-CO₂H) was purchased fromLaboTest (Niederschöna, Germany), and bovine serum albumin (BSA) waspurchased from Jackson ImmunoResearch Laboratories (West Grove, Pa.).(2-(2-(2-(2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)aceticacid was purchased from Prochimia (Gdansk, Poland). The glass microscopeslides and eight-well chamber slides were purchased from FisherScientific (Pittsburgh, Pa.). Gold electron microscopy grids (20 μmthickness, 50 μm bar width, and 283 μm hole width) were purchased fromElectron Microscopy Sciences (Hatfield, Pa.).

Preparation of Self-Assembled Monolayers (SAMs) on Gold. Gold Films Usedin the surface IR experiments were prepared on silicon wafers mounted ona rotating planetary in an electron beam evaporator (VES-3000-C, Tek-VacIndustries, Brentwood, N.Y.). The rotation of the substrates duringdeposition ensured that the gold films were deposited without apreferred direction of incidence. The silicon wafers were first coatedwith 100 Å of titanium at a rate of ˜0.2 Å/s to promote the adhesionbetween the silicon wafers and gold. Gold (thickness ˜2000 Å) was thendeposited at a rate of ˜0.2 Å/s. The substrates coated with gold filmswere cut into pieces (ca. 0.5×2.5 cm), rinsed with absolute ethanol, andthen dried under a stream of nitrogen. The slides were immersed in 0.1mM ethanolic solution of(2-(2-(2-(2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)aceticacid for 24 h. The resulting self-assembled monolayers (SAMs) werethoroughly rinsed with ethanol and dried using a gaseous stream ofnitrogen before use.

Preparation of Optical Cells. Glass Microscope Slides were Cleaned andfunctionalized with OTS according to the previously reported procedures.The OTS-coated glass slides were cut into pieces (ca. 5 mm×5 mm) and thepieces were fixed on the bottom of each well of an eight-well chamberslide with epoxy. Gold electron microscopy grids were then placed ontothe OTS-coated glass slides. The 5CB was dispensed onto the gridssupported on the OTS-coated glass slides, and the excess liquid crystalwas removed by using a capillary tube. Aqueous phosphate buffered saline(PBS) (10 mM phosphate, 120 mM NaCl, 2.7 mM KCl; pH 7.6) was quicklyintroduced into the well with a syringe. To prepare a mixed monolayer ofsurfactants at the aqueous-liquid crystal interface, the solution wasprepared by probe sonication (at 8 W for 5 min) of a mixture oftetraethylene glycol monotetradecyl ether and3,6,9,12,15-pentaoxanonacosanoic acid dissolved in the PBS (0.20 mM and0.80 mM, respectively). The PBS contacting with liquid crystal wasexchanged with the aqueous solution of surfactants. The aqueous solutionwas equilibrated with the interface of the liquid crystal for 1 h at rt.Throughout the experiment, aqueous solutions were always exchanged suchthat the meniscus did not fall below the liquid crystal interface toprevent the displacement of the liquid crystal from the grid.

Cross linking of Oligopeptides. The aqueous solution was exchanged fivetimes with the PBS (pH 7.6) before any other aqueous solution wasintroduced into the well. The solution of NHS (0.017 g) and EDC (0.115g) in 3.0 mL of the PBS was introduced into the well and equilibratedwith the mixed monolayer for 1.5 h at rt. Then, the NHS/EDC solution wasreplaced with the solution of 17-amino acid oligopeptide in the PBS(0.20 mM). After 1.5 h at rt, the oligopeptide solution was replacedwith the solution of ethanolamine in the PBS (20 mM) to quench remainingNHS-activated carboxylic acid groups. The ethanolamine solution wasallowed to be reacted for 30 min at rt before the protein solution ofinterest is introduced. The solutions of trypsin, trypsin and trypsininhibitor, and BSA were prepared in 30 mM HEPES buffer (pH 8.0) with 20mM CaCl₂. Before the introduction of the protein solutions, the aqueoussolution was exchanged five times with the HEPES buffer.

Characterization. The optical images of liquid crystals were monitoredbetween crossed polarizers of an optical microscope (BX60, Olympus,Tokyo, Japan). The optical images were captured using a digital camera(Olympus C-4000 Zoom) with consistent settings of the microscope (50% ofmaximum intensity, 10% open aperture, 4× magnification) and camera(f-stop of 2.8 and shutter speed of 1/320s). IR measurements wereperformed using a Nicolet Magna-IR 860 Fourier transform spectrometerequipped with a liquid nitrogen cooled mercury-cadmium-telluride (MCT)detector, a photoelastic modulator (PEM-90, Hinds Instruments,Hillsboro, Oreg.), and a synchronous sampling demodulator (SSD-100, GWCTechnologies, Madison, Wis.). The polarized light was reflected from thegold films at an angle of incidence of 83°.

Results and Discussion

Cross-linking of Oligopeptides to NHS-activated Carboxylic Acid Groups.Before describing the immobilization of oligopeptides at anaqueous-liquid crystal interface, the inventors first investigated thecovalent coupling of primary amine groups of the oligopeptide toNHS-activated carboxylic groups on a solid surface. Past studies showedthat EDC reacts with carboxylic acid groups to form O-acylisoureaintermediate that readily reacts with a primary amine group but alsoundergoes fast hydrolysis. Therefore, the carboxylic acid groups areactivated with EDC in the presence of NHS to form N-succinimidyl estersthat also quickly react with a primary amine group. Our group hasreported the coupling of lysine residues of a protein ribonuclease A(RNase A) to NHS-activated carboxylic acid groups on a mixedself-assembled monolayer (SAM). Luk and coworkers havepreviously-described the covalent coupling of RNase A containing 10lysine residues and a primary amine group at the N-terminal with aNHS-activated surface and their binding ability by using ellipsometryand the orientational behavior of liquid crystals. Here, the inventorsinvestigated the covalent coupling of the 17-amino acid oligopeptidethat will be used for our further experiments with a NHS-activatedsurface by polarization modulation infrared reflection-absorptionspectroscopy (PM-IRRAS). The 17-amino acid oligopeptide possess twolysine residues and a primary amine group at the N-terminal, and thuscould form covalent bonds to NHS-activated carboxylic acid groupsimmobilized on a surface.

In order to investigate the coupling by PM-IRRAS, the inventors preparedthe CO₂H-terminated surface by self-assembly of(2-(2-(2-(2-(2-(2-(2-(11-mercaptoundecyloxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)ethoxy)acetic acid on gold. FIG. 14 a shows thePM-IRRAS spectrum of the CO₂H-terminated SAM in the carbonyl stretchingregion. The inventors assigned the peak at ˜1745 cm⁻¹ to the carbonylstretch of the free carboxylic acid groups and hydrogen-bondedcarboxylic groups on the surface. The inventors then immersed theCO₂H-terminated SAM in NHS/EDC solution (50 mM/200 mM) in phosphatebuffered saline (PBS) (10 mM phosphate, 120 mM NaCl, 2.7 mM KCl; pH 7.6)for 1 h at rt. The SAM was thoroughly washed with deionized water andethanol, and dried under a stream of nitrogen before PM-IRRASmeasurements. FIG. 14 b shows the PM-IRRAS spectrum of the NHS-activatedsurface. The inventors observed the appearance of three new bands at1745, 1785, and 1821 cm⁻¹. Based on the past study by Frey and Corn, theinventors assigned the peaks at 1745 and 1785 cm⁻¹ to asymmetric andsymmetric stretch of the NHS carbonyls, respectively. They also assignedthe peak at 1821 cm⁻¹ to the carbonyl stretch of the NHS ester. To testthe coupling of the 17-amino acid oligopeptide with the NHS-activatedsurface, we immersed the NHS-activated SAM in a 0.20 mM aqueous 17-aminoacid oligopeptide solution in the PBS for 1 h at rt, and measure thePM-IRRAS spectrum of the surface. As shown in FIG. 14 c, the peakscorresponding to the NHS carbonyls and NHS ester disappeared and threenew bands at 1549, 1668, and 1724 cm⁻¹ appeared. The inventors assignedthe peak at 1549 cm⁻¹ to the NH bend of an amide, the peak at 1668 cm⁻¹to the amide carbonyl stretch, and the peak at 1549 cm⁻¹ to the carbonylstretch of remaining carboxylic acid groups or carboxylic acid groups inthe 17-amino acid oligopeptide. It was noted that the 17-amino acidoligopeptide contains many amide bonds and the PM-IRRAS spectra cannotdistinguish those amide bonds from the amide bonds formed fromNHS-activated carboxylic acid groups. Nevertheless, the disappearance ofthe peaks corresponding to the NHS groups suggests that the treatment ofthe NHS-activated surface with the 17-amino acid oligopeptide leads tothe conversion of NHS-activated carboxylic groups to amides. It cantherefore be concluded that the 17-amino acid oligopeptide formscovalent bonds with the NHS-activated carboxylic acid groups at aninterface.

Orientational Response of Liquid Crystal to Oligopeptide Cross-linking.The inventors investigated the orientational responses of liquidcrystals during immobilization of the oligopeptide at an aqueous-liquidcrystal interface. All optical images of liquid crystals presented inthis example were obtained between crossed polarizers of an opticalmicroscope. Before immersion under an aqueous solution, the 5CBcontacting with the OTS-coated glass slide and air appeared dark. Sincethe anchoring of the liquid crystal at the liquid crystal-OTS interfaceis homeotropic (perpendicular), the dark appearance indicates aperpendicular orientation of the liquid crystal at the air-liquidcrystal interface. The inventors first introduced phosphate bufferedsaline (PBS) (10 mM phosphate, 120 mM NaCl, 2.7 mM KCl; pH 7.6) to thesample using a syringe. The optical appearance of 5CB became brightindicating the orientational transition of liquid crystals fromperpendicular to planar or tilted alignment at the aqueous-liquidcrystal interface (FIG. 15 a). In order to prepare a mixed monolayer ofOH- and CO₂H-terminated surfactants at the liquid crystal interface, weprepared an aqueous solution of 0.20 mM tetraethylene glycolmonotetradecyl ether (C14EG4) and 0.80 mM3,6,9,12,15-pentaoxanonacosanoic acid (C14EG4-CO₂H) in PBS (pH 7.6).After removal of the PBS contacting with the liquid crystal, theinventors added the aqueous solution of mixed surfactants to the sample.Within 1 min of contact with the surfactant solution, the opticalappearance of 5CB became uniformly dark indicating the anchoring of 5CBperpendicular to both the aqueous-liquid crystal and liquid crystal-OTSinterfaces (FIG. 15 b), indicating the transfer of the surfactants ontothe aqueous-liquid crystal interface. Next, the inventors exchanged theaqueous solution with NHS/EDC solution (50 mM/200 mM) in PBS (pH 7.6) inorder to activate the carboxylic acid groups at the interface. Duringthe NHS-activation for 1.5 h at rt, the optical appearance of 5CBremained dark indicating homeotropic alignment of 5CB (FIG. 15 c). Theresult suggests that the NHS-activation of carboxylic acid groups at theliquid crystal interface does not perturb the orientation of liquidcrystals. The inventors then replaced the NHS/EDC solution with 0.20 mM17-amino acid oligopeptide solution in PBS (pH 7.6) and characterizedthe optical appearance of the liquid crystal by using polarized lightmicroscopy (FIG. 15 d). During 1.5 h of contact with the oligopeptidesolution, the optical appearance of 5CB became bright and colorful. Thebright image of 5CB indicates the anchoring transition of liquidcrystals from homeotropic to tilted alignment. It appears that thestructural strain inflicted by the 17-amino acid oligopeptideimmobilized at the aqueous-liquid crystal interface could cause changesin the orientation of alkyl chains in the monolayer, leading to theorientational transition of liquid crystals to tilted alignment.

In order to understand the orientational response of liquid crystals tothe immobilization of the 17-amino acid oligopeptide, the inventorscarried out several control experiments. First, an aqueous solution ofthe 17-amino acid oligopeptide was introduced to the mixed monolayerconsisting of OH- and CO₂H-terminated surfactants without NHS-activationof the carboxylic acid groups. Without NHS-activation, the primary aminegroups of the oligopeptide cannot form covalent bonds with thesurfactants. After 1.5 h of contact with the 17-amino acid oligopeptide,the optical appearance remained dark indicating homeotropic alignment of5CB at the aqueous-liquid crystal interface (FIG. 16 a). The resultsuggests that the orientation of 5CB is not perturbed without theformation of covalent bonds between the monolayer and the oligopeptide.Next, after the NHS activation of mixed monolayer, the inventorsintroduced three different compounds that commonly possess primary aminegroups. The aqueous solution of in PBS (pH 7.6) was equilibrated withthe NHS-activated monolayer for 1.5 h at rt. During incubation, theoptical appearance of 5CB also remained homogeneously black (FIGS. 16b-d). Although the primary amine groups of ethanolamine, DL-lysine, andpolylysine are likely to form covalent bonds with the NHS-activatedcarboxylic groups, the immobilization of those compounds did not perturbthe anchoring of the liquid crystals. It therefore appears that theprimary amine groups specifically constrained along the backbone areessential to cause changes in the structure of the mixed monolayer andthus the anchoring transition of liquid crystals.

Selective Detection of Enzymatic Cleavage. The results of controlexperiments described above suggest that the tilted orientation of 5CBat the aqueous-liquid crystal interface is likely to be caused bystructural strain introduced by the immobilized oligopeptides. It wastherefore believed that the orientation of 5CB at theoligopeptide-immobilized interface could be altered by contacting withan enzyme that cleaves the oligopeptide substrate because the enzymaticcleavage can release the structural strain inflicted by theoligopeptide. Consequently, the inventors then used trypsin that cancleave the 17-amino acid oligopeptide at four different locations.Trypsin selectively cleaves peptide bonds after (on the C-terminal sideof) lysine (K) and arginine (R). The optimum pH for trypsin is about 8,and the addition of moderate amounts of CaCl₂ (20 mM) can maximize thetrypsin activity and stabilize the protease. The inventors thereforeprepared a 200 nM trypsin solution in 30 mM HEPES buffer (pH 8.0)containing 20 mM CaCl₂ and introduced the solution to theoligopeptide-immobilized liquid crystal interface. Within 1 min ofcontact with the trypsin solution, the bright image of 5 CB exhibitedchanges in the interference colors from red, green, and yellow to yellowand gray, indicating decrease in tilt (relative to the surface normal)of 5CB at the aqueous-5CB interface (FIG. 17). After 5 min of contactwith the trypsin solution, the optical appearance of 5CB becamehomogeneously black indicating the anchoring transition of 5CB tohomeotropic alignment. To prove the proposition that this orientationaltransition of 5CB could be caused by the enzymatic cleavage of theoligopeptides, the inventors used the trypsin-chymotrypsin inhibitorfrom Glycine max (Bowman-Birk inhibitor). Before the addition of trypsinsolution to the sample, the trypsin-chymotrypsin inhibitor was added tothe trypsin solution to give 1:2 weight ratio of trypsin to theinhibitor. After incubated for 10 min at rt, the aqueous solution oftrypsin and inhibitor was introduced to the sample. In contrast totrypsin, the mixture of trypsin and the inhibitor did not show theorientational transition of 5CB to homeotropic alignment after 1 h ofincubation (FIG. 17). The inventors also performed a control experimentwith BSA that cannot cleave the 17-amino acid oligopeptide. BSA also didnot exhibit the anchoring transition of 5CB (FIG. 17).

The results, as a whole, suggest that the orientational transitionobserved with trypsin treatment arises from the enzymatic cleavage ofthe oligopeptide rather than nonspecific binding or other interactions.Therefore, the immobilization of oligopeptides at aqueous-liquid crystalinterface introduces structural strain into the monolayer, and therelease of the structural strain by enzymatic cleavage could causechanges in monolayer structure and anchoring of liquid crystals. Thestrategy reported in this example can be used to selectively reportenzymatic activities when an appropriate oligopeptide substrate isimmobilized.

Example 6 Preparation of a Device Containing a Polymerized Membrane atthe Interface between a Nematic Liquid Crystal and an Aqueous Phase—thePolymerized Membrane Containing the Antibody for Botulinum Neurotoxin

This example describes the polymerization of antibodies with adipoylchloride at interfaces between thermotropic liquid crystals and aqueousphases to facilitate the detection of botulinum neurotoxin according tothe present invention. Specifically, antibodies against botulinum toxinare polymerized with adipoyl chloride as a cross linker at the interfacebetween liquid crystals and aqueous phases. The adipoyl chloride isadded to the liquid crystal 4-cyano-4′-pentylbiphenyl (5CB) andhomogeneously mixed by sonication. The composition of the mixture isabout 1.5 wt. % adipoyl chloride in 5CB. The mixture is deposited intothe pores of gold TEM grids supported on octyltrichlorosilane(OTS)-coated glass slides. The antibody in PBS at a concentration of 1micromolar is introduced to the sample. After polymerization for 2 hoursat room temperature, the antibody solution is removed and the sample isrinsed with a slightly basic water (pH 8) more than five times. Thuslyprepared, a sample containing botulinum toxin may now be introduced tothe polymerized antibody at the aqueous phase/LC interface to facilitatedetection of the toxin by a change in orientation of the LC.

Example 7 Preparation of a Device Containing a Polymerized Membrane atthe Interface between a Nematic Liquid Crystal and an Aqueous Phase—thePolymerized Membrane Containing the Phospholipase A₂ Substratestearoyl-2-acyl-3-sn-glycerophosphorylethanolamine

This example describes the polymerization of substrates forphospholipase A₂ with adipoyl chloride at interfaces betweenthermotropic liquid crystals and aqueous phases. The substrate ispolymerized with adipoyl chloride as a cross linker at the interfacebetween liquid crystals and aqueous phases. The adipoyl chloride isadded to the liquid crystal 4-cyano-4′-pentylbiphenyl (5CB) andhomogeneously mixed by sonication. The composition of the mixture isabout 1.5 wt. % adipoyl chloride in 5CB. The mixture is deposited intothe pores of gold TEM grids supported on octyltrichlorosilane(OTS)-coated glass slides.Stearoyl-2-acyl-3-sn-glycerophosphorylethanolamine at a concentration of1 millimolar is introduced and, after polymerization for 2 hours at roomtemperature, the stearoyl-2-acyl-3-sn-glycerophosphorylethanolaminesolution is removed and the sample is rinsed with a slightly basic water(pH 8) more than five times. Thusly prepared, a sample containing anenzyme having phospholipase A₂ activity may now be introduced to thepolymerized tearoyl-2-acyl-3-sn-glycerophosphorylethanolamine at theaqueous phase/LC interface to facilitate detection of the enzyme by achange in orientation of the LC (described further in Example 9).

Example 8 Selective Detection of Antibody for Botulinum Neurotoxin

A polymerized membrane of botulinum neurotoxin is prepared at theaqueous-5CB interface by addition of 1 micromolar botulinum neurotoxinto the aqueous phase, and adipoyl chloride to the liquid crystal.Following polymerization of botulinum neurotoxin, the aqueous solutionis replaced by a solution containing antibodies to botulinum neurotoxin.After binding of the antibodies to the polymerized membrane of botulinumneurotoxin, DLPC is added to the aqueous phase containing theantibodies. To a second aqueous phase contacting a botulinum neurotoxinmembrane/liquid crystal that has not been contacted with a sample, DLPCis also added. The time at which the order of the LC changes followingaddition DLPC is different in the presence/absence of the antibody, thusallowing the presence of antibody in a sample to be detected.

Example 9 Selective Detection of Phospholipase A₂ (PLA2) ActivityEnzymatic Cleavage

A membrane containing a polymerized substrate for PLA2 is prepared asdescribed in example 7. A sample containing PLA2 and calcium ions isadded to the aqueous phase and incubated against the membrane.Observation of the optical appearance of the liquid crystal reveals achange in order of the liquid crystal in the presence of the PLA2. In acontrol experiment, PLA2 is added to the aqueous solution in the absenceof Ca2+ ions but in the presence of EDTA. In this experiment, there isno observable change in the optical appearance of the liquid crystal.This example demonstrates the use of the invention to detect theenzymatic activity of PLA2 using a cross-linked membrane containing asubstrate for PLA2.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific materials and methods described herein. Such equivalents areconsidered to be within the scope of this invention and encompassed bythe following claims.

1. A liquid crystal device for detecting the presence of a bioagent in asample, comprising: (a) a liquid crystal; (b) an aqueous phasepositioned such that an interface exists between the liquid crystal andsaid aqueous phase; and (c) a membrane located at the interfacecontaining a polymerized target of a bioagent, said polymerized targetcomprising a target of the bioagent covalently cross-linked to a polymernetwork, wherein interaction of said bioagent with the polymerizedtarget causes an orientation change in said liquid crystal therebyindicating the presence of the bioagent in the sample.
 2. The liquidcrystal device according to claim 1 wherein said bioagent is an enzymeand the target is a substrate for the enzyme.
 3. The liquid crystaldevice according to claim 1 wherein said bioagent is an antibody and thetarget is an antigen recognized by said antibody.
 4. The liquid crystaldevice according to claim 1 wherein said bioagent is an antigen and thetarget is an antibody that recognizes said antigen.
 5. The liquidcrystal device according to claim 1 wherein the polymer networkcomprises a non-target molecule that is cross-linked to said target. 6.The liquid crystal device according to claim 5, wherein the non-targetmolecule is a polymer.
 7. The liquid crystal device according to claim1, wherein the aqueous phase comprises an enhancing agent that enhancesthe change in orientation of the liquid crystal upon interaction of thebioagent and target.
 8. The liquid crystal device according to claim 7wherein the enhancing agent is dilauroyl phosohatidylcholine (DLPC). 9.The liquid crystal device according to claim 1, further comprising across-linking agent.
 10. A liquid crystal device for detecting thepresence of a bioagent in a sample, comprising: (a) a liquid crystal;(b) an aqueous phase positioned such that an interface exists betweenthe liquid crystal and said aqueous phase; and (c) a polymerizedmembrane comprising a polymer cross-linked to a target of a bioagent,said polymerized membrane located at the interface between the liquidcrystal and said aqueous phase wherein interaction of said bioagent withthe target causes an orientation change in said liquid crystal therebyindicating the presence of the bioagent in the sample.